表观遗传学在中枢神经系统肿瘤和神经胶质瘤的分层中发挥着至关重要的作用。研究神经胶质瘤中的分泌性卷曲相关蛋白 4 (SFRP4) 的重要性在于改善弥漫性神经胶质瘤甲基化谱。在这里，我们检测了WHO 2-4级弥漫性胶质瘤中SFRP4启动子的甲基化状态及其蛋白表达水平。通过免疫组织化学检测SFRP4表达并进行半定量评估。在肿瘤热点区域，使用ImageJ（美国国立卫生研究院）测定200个细胞中的蛋白表达强度。免疫阳性的评估基于 IRS 评分（免疫反应评分）。通过甲基化特异性 PCR (MSP) 在 51 个弥漫性神经胶质瘤样本和适当的对照中检查启动子甲基化。分离的 DNA 经过亚硫酸氢盐转化处理，然后用于 MSP。检索公共数据库（cBioPortal、COSMIC和LOVD）以证实结果。胶质母细胞瘤中86.7%的样本中SFRP4蛋白表达非常弱或不存在，13.3%的样本中表达中等，而没有观察到强表达。星形细胞瘤级别的增加导致SFRP4蛋白减少(p = 0.008)，表明其在Wnt信号传导中拮抗作用的丧失。 16.3%的病例发现SFRP4基因启动子甲基化。与 3 级星形细胞瘤 (p = 0.004) 和胶质母细胞瘤 (p < 0.001) 相比，2 级星形细胞瘤的甲基化病例明显更多，这可能表明 2 级星形细胞瘤存在甲基化的时间生态位。此外，在甲基化样本中 SFRP4 的表达水平较高SFRP4 启动子在非甲基化情况下较低或缺失（Pearson's R = -0.413；p = 0.003）。我们还研究了 SFRP4 变化与关键 Wnt 调节因子 GSK3β 和 DKK3 的关联，并建立了 SFRP4 和 GSK3β 甲基化之间的正相关性（Pearson's R = 0.323；p = 0.03）。此外，SFRP4 表达与未甲基化 DKK3 相关（卡方 = 7.254；p = 0.027），表明 Wnt 信号拮抗剂与负调节因子的去甲基化相关。该研究有助于认识弥漫性神经胶质瘤中表观遗传变化的重要性，表明恢复 SFRP4蛋白质具有作为治疗途径的潜力。胶质母细胞瘤中 SFRP4 的表达减少，不遵循启动子甲基化模式，表明另一种机制，可能是全局甲基化，可以在更高级别关闭 SFRP4 表达。版权所有 © 2024 Kafka、Pećina-Šlaus、Drmić、Bukovac、Njirić、Žarković 和 Jakovčević。

Epigenetics play a vital role in stratifying CNS tumors and gliomas. The importance of studying Secreted frizzled-related protein 4 (SFRP4) in gliomas is to improve diffuse glioma methylation profiling. Here we examined the methylation status of SFRP4 promoter and the level of its protein expression in diffuse gliomas WHO grades 2-4.SFRP4 expression was detected by immunohistochemistry and evaluated semi-quantitatively. In the tumor hot-spot area, the intensity of protein expression in 200 cells was determined using ImageJ (National Institutes of Health, United States). The assessment of immunopositivity was based on the IRS score (Immunoreactivity Score). Promoter methylation was examined by methylation specific-PCR (MSP) in fifty-one diffuse glioma samples and appropriate controls. Isolated DNA was treated with bisulfite conversion and afterwards used for MSP. Public databases (cBioPortal, COSMIC and LOVD) were searched to corroborate the results.SFRP4 protein expression in glioblastomas was very weak or non-existent in 86.7% of samples, moderate in 13.3%, while strong expression was not observed. The increase in astrocytoma grade resulted in SFRP4 protein decrease (p = 0.008), indicating the loss of its antagonistic role in Wnt signaling. Promoter methylation of SFRP4 gene was found in 16.3% of cases. Astrocytomas grade 2 had significantly more methylated cases compared to grade 3 astrocytomas (p = 0.004) and glioblastomas (p < 0.001), which may indicate temporal niche of methylation in grade 2. Furthermore, the expression levels of SFRP4 were high in samples with methylated SFRP4 promoter and low or missing in unmethylated cases (Pearson's R = -0.413; p = 0.003). We also investigated the association of SFRP4 changes to key Wnt regulators GSK3β and DKK3 and established a positive correlation between methylations of SFRP4 and GSK3β (Pearson's R = 0.323; p = 0.03). Furthermore, SFRP4 expression was correlated to unmethylated DKK3 (Chi square = 7.254; p = 0.027) indication that Wnt signaling antagonist is associated to negative regulator's demethylation.The study contributes to the recognition of the significance of epigenetic changes in diffuse glioma indicating that restoring SFRP4 protein holds potential as therapeutic avenue. Reduced expression of SFRP4 in glioblastomas, not following promoter methylation pattern, suggests another mechanism, possible global methylation, that turns off SFRP4 expression in higher grades.Copyright © 2024 Kafka, Pećina-Šlaus, Drmić, Bukovac, Njirić, Žarković and Jakovčević.